The biosynthesis and breakdown of glutathione, G, and ophthalmic acid, OA, in the bovine and rodent lens are being evaluated. The investigation is focussed on the transport of potential precursors, and their subsequent chemical conversion and incorporation into the tripeptides. A major outstanding problem involves the transport and intracellular oxidation/reduction of cystine and cysteine-candidates for the central residue of G. Sulfhydryl groups will be reacted with ICH2CONH2 to yield stable derivatives for fractionation and identification by ion exchange chromatography. Rates of incorporation into G are determined with a stream splitter by a combination of amino acid analysis and estimation of radioactivity passing through a flow cell packed with anthracene crystals. The rates of incorporation of all three residues of G and OA will be compared in the calf lens to provide additional information on the two enzymic reactions involved in the synthesis. The normally low lenticular levels of the central amino acids, cysteine and gamma-amino-n-butyric acid, gamma-AB, may be rate- limiting. Therefore, the effect of elevated levels of these amino acids on rates of peptide formation will be examined. (This effect can readily be achieved by incubation in the presence of cysteine, cystine, or gamma-AB). If gamma-glutamylsynthetase is responsible for synthesis of both peptides, competitive effects should be observed when both cysteine and gamma-AB are present in the cells of the lens. This competition may be studied by pre-incubating lenses in the presence of the stable isotopic form of one of the amino acids, followed by incubation in the presence of the radioactive form of the other. Attempts will be made to investigate the catabolism of G by incubation of lenses in the presence of methionine sulfoximine, an inhibitor of gamma-glutamy synthetase. Changes in level of G will be followed by colorimetry with 5,5'-dithiobis-(2-nitrobenzoic acid). Radioactive cystine and glutamine will be used as tracers to mark the presence of catabolites in ion exchange chromatography.